Separations using high performance liquid chromatography (HPLC) rely on the fact that a number of component solute molecules in a flowing stream of a fluid percolate through a packed bed of particles, known as the stationary phase. This allows particles to be efficiently separated from one another. Each component has a different affinity for the stationary phase, leading to a different rate of migration and exit time for each component from the column. The separation efficiency is determined by the amount of spreading of the solute band as it traverses the column.
Plate theory is commonly used to describe the passage of a solute through a chromatographic column and the band broadening. Plate theory explains band broadening in terms of a number of rate factors. For instance, separations may be considered to be made in a plurality of connected, equal, discrete, hypothetical steps, each volume of which contains both stationary and moving phases and in each of which complete equilibrium is established. Each such stage is called a “theoretical” plate. In such cases the number of theoretical plates in the column is calculated from the degree of separation. The length of the column is important to this calculation in relation to the theoretical plates. The length of the column per calculated theoretical plate is called the “height equivalent to a theoretical plate” or H, and is a measure of the phenomenon of band broadening.
Band broadening is important to separations and is indicative of the quality of the separation. For instance, generally speaking the broader the bands in the separation the worse the separation or column efficiency.
Separations are also based on the relationship between the phases. In chromatography, one phase is stationary and the other phase is mobile. The mobile phase moves past the stationary phase at a relatively fast rate so that complete equilibrium is, in fact, not attained between the two phases. This must be considered when performing separations to avoid peak broadening and to obtain clean and efficient separations.
In applying plate theory to chromatographic columns, all of the solute is assumed to be present initially in the first plate volume of the column. Dispersion is based on the distribution coefficient. In this instance, the distribution coefficient is constant for the solute concentrations encountered, and the solute rapidly distributes itself between the two phases in each plate volume.
Columns that provide minimum peak broadening are indicative of clean separations and are desirable for HPLC systems and processes. The nature of the packing put into the column and manner in which the column is packed, are all of high importance in getting clean and effective separations of sample components. It is, therefore, desirable to minimize the various processes that determine relative band broadening with deleterious effects on column performance. The effect of each of these processes on the column plate height H can be related by rate theory to such experimental variables as mobile-phase velocity u, packing particle diameter dp, and the solute diffusion coefficient in the mobile phase. The major band broadening processes in HPLC contributing to height equivalent to a theoretical plate, H, are generally considered by the Van Deemter equation. In addition, this equation can be simplified to relate the three important variables of particle size, fluid velocity and diffusion coefficient. In this reduced equation, as the particle size increases the efficiency should decrease. As the fluid velocity increases, the equation predicts that the mass transfer term will dominate the efficiency with a deterioration proportional to the product of the velocity and particle diameter. It will be appreciated that minimization of band broadening is desirable to insure that one obtains optimum separation of solutes, in analytical chromatography, product purity, and preparative chromatography.
However, the advantages obtained from smaller particles in terms of column efficiency must be offset by the disadvantages of higher back pressure, which include increased instrument cost and reduced reliability caused by the higher system stresses. Furthermore, it is normally asserted that ideally, particles used in liquid chromatography columns should be monodisperse, and have as narrow a particle size distribution as possible. For example, Dewaele and Verzele disclose (J. Chromatography, 260 (1983) 13-21) that blends of irregular shaped materials with different particle sizes yield packing with greater pressure than the corresponding equivalent mean particle size. The work by Dewaele and Verzele was restricted to two component blends. Each component of the blends having a monodisperse particle distribution and a mean for the distribution. In addition, the means of the particle size distributions differed from each other by greater than 40%. In other words, blends of particles that differed greatly in mean diameter where ineffective in lowering overall back pressure of the system.
It, therefore, would be desirable to provide a novel material or materials that would allow for efficient and effective separations, yet avoid the problem of increased back pressure and poor separations and efficiency.